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A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei.

Sebastian Shaw, Sebastian Knüsel, Sarah Hoenner, Isabel Roditi

Preprint posted on 9 May 2020 https://www.researchsquare.com/article/rs-21224/v1

A novel addition to the toolbox for genome editing Trypanosoma brucei.

Selected by Mariana De Niz

Categories: molecular biology

Background

The establishment of genome editing by CRISPR/Cas9 has greatly increased the ease across fields of research, with which mutant organisms can be generated. For Trypanosoma brucei parasites CRISPR/Cas9 has greatly facilitated the genetic modification (knock outs and tagging) of parasites, as it makes possible the deletion of two copies of non-essential genes in a single round of transfection. The most widely used system for T. brucei, uses cell lines which express Cas9 and T7RNA polymerase (T7RNAP). However, this limits the method to the use of such lines. In their work, Shaw et al introduce a novel system allowing genome editing of any T. brucei cell line (1).

Figure 1. Plasmid map of pAi1C9, and tagged trypanosomes using the novel genome editing strategy.

 

Key findings and developments

            The authors generated a novel sequential transfection expression system which allows transient expression of Cas9, T7RNAP and guide RNAs, by modification of a plasmid currently used for CRISPR/Cas9-based editing in T. brucei (pTB011) (2). The new plasmid (pAi1C9) allows genome editing of any cell line, without the need for stable integration of the Cas9 and T7RNAP genes. To test the functionality of the plasmid, the authors successfully generated various mNG-tagged parasites as well as knock-out T. brucei lines of non-essential genes. The authors discuss as a limitation of this system, that it gives rise to fewer clones than cell lines stably transformed with T7RNAP and Cas9. As advantages, the authors highlight that this system can be applied to any trypanosome cell line, and circumvents possible Cas9 toxicity.

 

What I like about this preprint

I like that it constitutes a significant and valuable improvement for the generation of mutant Trypanosoma parasites. In some fields, the introduction of novel methods takes a long time, however this improvement comes relatively soon after the introduction of CRISPR/Cas9 altogether to the toolbox relevant for this parasite. It will likely facilitate the range of modification possible, throughput, and ultimately study of important genes in Trypanosoma brucei.

 

Open questions

  1. Why does pAi1C9 result in fewer clones than using cell lines stably transformed with T7RNAP and Cas9 genes, and how can you overcome this limitation in your current system?
  2. Beyond the number of clones obtained, do you think characterization of parasite mutants generated using transient vs. stable transfection might show different results for some targets?
  3. As future directions, what tools do you envisage would be useful for the genetic modification and phenotypic characterization of brucei mutants? For instance, in Plasmodium research, the use of barcoding technology (3) was revolutionary. Can your strategy be coupled to this type of high throughput phenotypic characterization?
  4. You mention that your procedure could be adapted for targeting RNAs or epigenetic mutations. This would be highly relevant to the field of brucei. Do you plan to use it in this context soon?
  5. Is your system translatable to other Trypanosoma species, such as T. congolense, T. vivax, T. cruzi? If not, and it would be a useful advance, do you envisage adapting it to study these species too?

 

References

  1. Shaw S, Knüsel S, Hoenner S, Roditi I, A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei, BMC research notes,
  2. Beneke T, Madden R, Makin L, Valli J, Sunter J, Gluenz E, A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. R Soc Open Sci, 2017
  3. Gomes AR, Bushell E, Schwach F, Girling G, Anar B, Quail MA, Herd C, Pfander C, Modrzynska K, Rayner JC, Billker O, A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite. Cell Host and Microbe, 2015

 

Acknoweldgements

I am very grateful to Isabel Roditi and Sebastian Shaw for their input and engaging in helpful discussion.

 

Posted on: 9 May 2020

doi: https://doi.org/10.1242/prelights.20342

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Author's response

Isabel Roditi and Sebastian Shaw shared

Open questions

1.Why does pAi1C9 result in fewer clones than using cell lines stably transformed with T7RNAP and Cas9 genes, and how can you overcome this limitation in your current system?

Because only about 10-20 % of the cells take up the plasmid during transient transfection.  It will be difficult to increase this using the current buffers and electroporators.

2.Beyond the number of clones obtained, do you think characterization of parasite mutants generated using transient vs. stable transfection might show different results for some targets?

We have no reason to expect this if the sgRNAs are the same and mutants are validated for correct integration.

3.As future directions, what tools do you envisage would be useful for the genetic modification and phenotypic characterization of T. brucei mutants? For instance, in Plasmodium research, the use of barcoding technology (3) was revolutionary. Can your strategy be coupled to this type of high throughput phenotypic characterization?

This is unlikely because of one important difference between the two parasites. Trypanosomes are diploid for most of their life cycle whereas Plasmodium is haploid.

4.You mention that your procedure could be adapted for targeting RNAs or epigenetic mutations. This would be highly relevant to the field of T. brucei. Do you plan to use it in this context soon?

We are concentrating on other projects for the moment, but will keep this in mind.

5.Is your system translatable to other Trypanosoma species, such as T.  congolense, T. vivax, T cruzi? If not, and it would be a useful advance, do you envisage adapting it to study these species too?

In principle it should be translatable and useful, but we are not working on this.  It could be that a simple promoter swap is enough, but it may also require using the intergenic regions from the corresponding parasite to ensure that mRNA processing happens correctly.

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